The long-term goal is to understand how initiation of DNA replication is performed in Escherichia coli and how it is regulated. Initiation of DNA replication is an obligatory event in the cell cycle of any organism, and understanding this complex process will tell us how a cell can control and modulate its passage through the cell cycle. The possibilities of revealing these control mechanisms is considered best by studying the cell that is best characterized biochemically, genetically, and physiologically, namely the bacterium Escherichia coli. In the long run, information about cell controls in prokaryotes may shed light on control mechanisms also in eukaryotes and contribute to our understanding of cell proliferation diseases such as e.g. cancer. Our approach will be to use molecular biological tools (gene cloning, DNA sequencing, in vitro assays) combined with use of a unique flow cytometer designed and constructed in our department. With this instrument we can measure, with high precision, the cell size and DNA, RNA or protein contents of individual bacterial cells. This enables us to closely monitor events such as initiation of DNA replication and cell division and to get data on the kinetics of DNA replication and mass increase not obtainable by other methods. We wish to study the role of the dnaA gene product in initiation of DNA replication. The dnaA protein is known to play a key role in this complex process, but its role in controlling or regulating initiation is no known. We also wish to investigate in detail the molecular interactions occurring in the region around the origin of replication and the significance of different DNA sequences for these interactions. In particular, we will study methylation of the adenines in GATC sequences within the origin and its effects on initiation.